Fluorescence detection of glutathione reductase activity based on deoxyribonucleic acid-templated silver nanoclusters

Fluorescent silver nanoclusters stabilized by DNA (DNA-AgNCs) exhibit distinct response rates to thiol and disulfide. Glutathione reductase can catalyze the reduction of the oxidized glutathione (GSSG) quickly to reduced glutathione (GSH) in the presence of β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH). Consequently, DNA-AgNCs can serve as a new fluorescent platform for assaying the glutathione reductase (GR) activity. This newly proposed assay has a high sensitivity and a good selectivity toward GR. The GR activity can be detected in the range of 0.2–2.0 mU mL⁻¹ with a minimum detectable concentration of 0.2 mU mL⁻¹. Pepsin, lysozyme, trypsin, avidin, thrombin, myoglobin, and BSA have little effect on the fluorescence intensity of DNA-AgNCs. The GR activity assay is successfully used to monitor the inhibition of GR activity by a typical inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea.     

Proteomic and redox‐proteomic study on the role of glutathione reductase in human lung cancer cells

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR‐modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1‐0shControl, and its GR‐knockdown derivative, CL1‐0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin‐1 overexpression in the air‐exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR‐modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox‐proteomic analysis used to investigate the role of GR in a mammalian cell model.     

Purification of glutathione reductase from chicken liver and investigation of kinetic properties

Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K _M and V _max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.     

1,3-Oxazole derivatives of cytisine as potential inhibitors of glutathione reductase of Candida spp.: QSAR modeling,docking analysis and experimental study of new anti-Candida agents

Natural products as well as their derivatives play a significant role in the discovery of new biologically active compounds in the different areas of our life especially in the field of medicine. The synthesis of compounds produced from natural products including cytisine is one approach for the wider use of natural substances in the development of new drugs. QSAR modeling was used to predict and select of biologically active cytisine-containing 1,3-oxazoles. The eleven most promising compounds were identified, synthesized and tested. The activity of the synthesized compounds was evaluated using the disc diffusion method against C. albicans M 885 (ATCC 10,231) strain and clinical fluconazole-resistant Candida krusei strain. Molecular docking of the most active compounds as potential inhibitors of the Candida spp. glutathione reductase was performed using the AutoDock Vina. The built classification models demonstrated good stability, robustness and predictive power. The eleven cytisine-containing 1,3-oxazoles were synthesized and their activity against Candida spp. was evaluated. Compounds 10, 11 as potential inhibitors of the Candida spp. glutathione reductase demonstrated the high activity against C. albicans M 885 (ATCC 10,231) strain and clinical fluconazole-resistant Candida krusei strain. The studied compounds 10, 11 present the interesting scaffold for further investigation as potential inhibitors of the Candida spp. glutathione reductase with the promising antifungal properties. The developed models are publicly available online at http://ochem.eu/article/120720 and could be used by scientists for design of new more effective drugs.     

Using Homology Modeling, Molecular Dynamics and Molecular Docking Techniques to Identify Inhibitor Binding Regions of Somatostatin Receptor 1

The G protein coupled receptor(GPCR), one of the members in the superfamily, which consists of thousands of integral membrane proteins, exerts a wide variety of physiological functions and responses to a large portion of the drug targets. The 3D structure of somatostatin receptor 1(SSTR1) was modeled and refined by means of homology modeling and molecular dynamics simulation. This model was assessed by Verify-3D and Vadar, which confirmed the reliability of the refined model. The interaction between the inhibitor cysteamine, somatostatin(SST) and SSTR1 was investigated by a molecular docking program, Affinity. The binding module not only showed the crucial residues involved in the interaction, but also provided important information about the interaction between SSTR1 on the one hand and ligands on the other, which might be the significant evidence for the structure-based design.     

Homology modeling, agonist binding site identification, and docking in octopamine receptor of Periplaneta americana

AY333178 (from Periplaneta americana, 628 AAs) was selected as a target octopamine receptor (OAR) class OAR2 for this study using Discovery Studio (DS Modeling1.1/1.2, Accelrys Inc.). Blast similarity search was performed and identified that AY333178 contains N-terminal domain of GPCR. Based upon Blast and Pfam results, Rhodopsin 1U19 (protein data bank) was considered as an ideal homologue and used as a template for homology modeling due to its higher X-ray resolution at 2.2A. Sequence alignment between AY333178 and 1U19 was done using Align123 followed by a manual modification. The final alignment was carefully evaluated and evidenced to be matching the conserved residue data for class A GPCR fairly well. The 3D model of AY333178 was generated with MODELER, and further refined using CHARMm. Superimposition of the model was done over the template 1U19. Two fairly consistent profiles were observed demonstrating AY333178 model was reasonable and could be employed for the further docking study. Agonist docking into OAR2 model was done using LigandFit. The superimposition of two top poses of representative agonists was performed with a soft surface generated. Those models are considered to be used in designing new leads for hopefully more active compounds. Further research on the comparison of models for the agonists may elucidate the mechanisms of OAR2-ligand interactions.     

A first-principle study of the stability and electronic properties of halide inorganic double perovskite Cs2PbX6 (X = Cl,I) for solar cell application

The problem of lead toxicity in perovskites materials that are currently performing with the most efficiency can be partially solved by choosing double perovskites compounds Cs₂PbX₆ (X = Cl,I), which have considerably reduced lead contents. These materials are slightly more stable, and substituting Cl and I with Br in small percentages further improves their mechanical stability and electronic properties. In this study, the properties of these promising materials were investigated in their pure and mixed forms.     

靛玉红类CDK1抑制剂的同源模建、分子对接及3D-QSAR研究

细胞周期蛋白依赖性激酶1的异常表达会导致G2期的停滞及多种肿瘤的发生,故CDK1近年来已成为一个理想的治疗靶点. 本文以细胞分裂调控蛋白2的同源体为模板,同源模建了CDK1的结构,并与靛玉红类小分子抑制剂进行分子对接. 分别运用三种叠合方法进行分子叠合,并在此基础上采用Sybyl 7.1中的比较分子场分析(CoMFA)模块及Discovery Studio 3.0中的三维定量构效关系(3D-QSAR)模块(以下简称为DS)分别建立了3D-QSAR模型. 其中,将分子对接叠合与公共骨架叠合联合运用的叠合方法所得3D-QSAR模型的评价参数是最佳的(CoMFA：q²=0.681,r²=0.909,r_pred.²=0.836; DS：q²=0.579,r²=0.971,r_pred.²=0.795,其中q²为交叉验证系数,r²为非交叉验证系数). 本文的研究结果在对靛玉红类小分子进行结构修饰设计出新的CDK1抑制剂方面,可提供重要的理论基础.     

α2A肾上腺素受体的同源模建及与Yohimbine的对接研究

通过对比多个与α2A-肾上腺素受体同属G-蛋白偶联受体的视紫红质蛋白序列,选择以相似性最大的牛视紫红质蛋白为模板,同源模建了α2A-肾上腺素受体的跨膜结构,并在结构中找到了体积为0.090 nm3,已被报导的活性残基包围的活性位点.运用分子力学与动力学方法研究了此结构突变前后与抑制剂Yohimbine的对接情况,得到了与文献报道相吻合的结果.同时对接研究结果发现,在α2A-肾上腺素受体的结合位点周围的一个由色氨酸和两个苯丙氨酸组成的局部疏水区对抑制剂有稳定作用,并且天冬氨酸113作为氢键受体也对稳定抑制剂有重要作用.     

Modeling the framework stability and catalytic activity of pure and transition metal-doped zeotypes

We present a thorough computational study of transition metal-doped zeolite and aluminophosphate (AlPO) frameworks. The structural and electronic chemistry of the dopants is examined with ab initio quantum mechanical calculations, and the results correlated with the Brønsted and Lewis acid strength, and with the redox potential of the dopant ions in the framework. The energetics of doping is provided, and is employed to analyze the mode of dopant incorporation, and its site ordering in the microporous framework. In total, 23 dopant ions are examined in the isostructural framework of chabasite and AlPO-34. These cover most of the isomorphous framework replacements known to occur experimentally, but also framework replacements that have not yet been achieved. In this case, ab initio modeling techniques are employed in a predictive way. Finally, we present a computational study of the alkene epoxidation on titanosilicates, that covers the whole catalytic cycle.     

同源模建和分子对接研究HDAC1/HDAC8的选择性

组蛋白去乙酰化酶(HDACs)是近年来治疗肿瘤的重要靶标之一.由于HDACs包含多种亚型,且各亚型的生理功能存在一定的差异,其选择性抑制剂的开发已成为当前的研发热点.我们通过同源模建的HDAC1结构,与已有的HDAC8晶体结构的活性位点进行比较分析,探讨了对两者选择性有重要影响的残基,为基于受体的选择性抑制剂研究提供重要信息.同时选择了52个HDAC抑制剂,分别建立了HDAC1、HDAC8的活性值与对接打分值的线性回归模型.所建的HDAC1和HDAC8的线性构效关系模型的非交叉验证系数R2分别为0.82和0.80,表明具有一定的统计学意义.利用所建模型对已设计合成的化合物进行了预测,预测结果对HDAC1、HDAC8选择性抑制剂的优化改造提供了一定的指导意义.     

Investigation of the effects of natural compounds isolated from Arum rupicola var. rupicola on glutathione reductase enzyme purified from bovine liver

Glutathione reductase (GR, E.C. 1.8.1.7), a flavoenzyme, is responsible for recycling of oxidized glutathione disulfide. This study was performed in two main sections. In the first GR was purified from bovine liver by affinity column chromatography and the purification rate and specific activity of the enzyme were calculated as 1832‐fold and 141 EU/mg protein, respectively. The subunit molecular weight of the enzyme was determined as 55 kDa by means of SDS–PAGE. The second section isolated natural components of Arum rupicola Boiss. var. rupicola using column chromatography. The isolation protocol for this plant was performed with a series of different‐sized columns with hexane–ethyl acetate. According to the thin‐layer chromatography plate, seven substances (R1–R7) were isolated. Our study's aim was to find new activators or inhibitors for GR activity. With this aim, all isolated substances were tested for GR activity. R6 showed competitive inhibition, while R4 had noncompetitive inhibition of GR activity. R1 played a role as an activator of GR activity. The inhibitory activity percentage vs. concentration graph was plotted. Values of IC₅₀ for R4 and R6 were calculated as 0.193 mg/mL and 3.98 μg/mL, respectively, from the equation of this graph.     

Molecular characterization,modeling and docking of CYP107CB2 from Bacillus lehensis G1, an alkaliphile

Cytochrome P450s are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. The diversity of chemical reactions catalyzed by cytochrome P450s has led to their increased demand in numerous industrial and biotechnology applications. A recent study showed that a gene sequence encoding a CYP was found in the genome of Bacillus lehensis G1, and this gene shared structural similarity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. The objectives of present study was to mine, for a novel CYP from a new isolate B. lehensis G1 alkaliphile and determine the biological properties and functionalities of CYP in this bacterium. Our study employed the usage of computational methods to search for the novel CYP from CYP structural databases to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. A computational homology model of the protein’s structure was generated and a docking analysis was performed to provide useful structural knowledge on the enzyme’s possible substrate and their interaction. Sequence analysis indicated that the newly identified CYP, termed CYP107CB2, contained the fingerprint heme binding sequence motif FxxGxxxCxG at position 336-345 as well as other highly conserved motifs characteristic of cytochrome P450 proteins. Using docking studies, we identified Ser-79, Leu-81, Val-231, Val-279, Val-383, Ala-232, Thr-236 and Thr-283 as important active site residues capable of stabilizing interactions with several potential substrates, including vitamin D₃, 25-hydroxyvitamin D₃ and 1α-hydroxyvitamin D₃, in which all substrates docked proximally to the enzyme’s heme center. Biochemical analysis indicated that CYP107CB2 is a biologically active protein to produce 1α,25-dihydroxyvitamin D₃ from 1α-hydroxyvitamin D₃. Based on these results, we conclude that the novel CYP107CB2 identified from B. lehensis G1 is a putative vitamin D hydroxylase which is possibly capable of catalyzing the bioconversion of parental vitamin D₃ to calcitriol, or related metabolic products.     

Modeling the inhibition of quadruple mutant <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> dihydrofolate reductase by pyrimethamine derivatives

Modeling studies were performed on known inhibitors of the quadruple mutant Plasmodium falciparum dihydrofolate reductase (DHFR). GOLD was used to dock 32 pyrimethamine derivatives into the active site of DHFR obtained from the x-ray crystal structure 1J3K.pdb. Several scoring functions were evaluated and the Molegro Protein-Ligand Interaction Score was determined to have one of the best correlation to experimental pK _i . In conjunction with Protein-Ligand Interaction scores, predicted binding modes and key protein-ligand interactions were evaluated and analyzed in order to develop criteria for selecting compounds having a greater chance of activity versus resistant strains of Plasmodium falciparum. This methodology will be used in future studies for selection of compounds for focused screening libraries.     

Modeling the polymerization behavior of vegetable‐oil‐derived macromonomers in solution by computer simulation

A computer simulation model was used to study the polymerization behavior of multifunctional, vegetable‐oil‐derived macromonomers. Mixtures of olefins (A) and acrylates (B) were initially randomly dispersed on a cubic lattice of size L³. Interactions between A, B, and the solvent sites were considered with respect to their relative proximity, mobility and some kinetics. The Metropolis algorithm was used to move each functional group (A and B). Stirred and equilibrated samples were prepared before reaction initiation. Reactions between the functional groups were implemented with a bonding probability k_αβ, which was subject to the availability of unsaturated bonds. The conversion factor, that is, the growth of A–B bonds, was analyzed for a range of polymer concentrations (p = 0.2–0.8) with different reaction probabilities (i.e., k_αβ). A stirred (nonequilibrium) sample did not allow sufficient time for the functional groups to arrange according to the interaction parameters. Therefore, the simulations were rerun with equilibrated samples and were found to be consistent with experimental observations. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 1164–1172, 2004     

Modeling of the catalytic core of Arabidopsis thaliana Dicer-like 4 protein and its complex with double-stranded RNA

Plant Dicer-like proteins (DCLs) belong to the Ribonuclease III (RNase III) enzyme family. They are involved in the regulation of gene expression and antiviral defense through RNA interference pathways. A model plant, Arabidopsis thaliana encodes four DCL proteins (AtDCL1-4) that produce different classes of small regulatory RNAs. Our studies focus on AtDCL4 that processes double-stranded RNAs (dsRNAs) into 21 nucleotide trans-acting small interfering RNAs. So far, little is known about the structures of plant DCLs and the complexes they form with dsRNA. In this work, we present models of the catalytic core of AtDCL4 and AtDCL4-dsRNA complex constructed by computational methods.We built a homology model of the catalytic core of AtDCL4 comprising Platform, PAZ, Connector helix and two RNase III domains. To assemble the AtDCL4-dsRNA complex two modeling approaches were used. In the first method, to establish conformations that allow building a consistent model of the complex, we used Normal Mode Analysis for both dsRNA and AtDCL4. The second strategy involved template-based approach for positioning of the PAZ domain and manual arrangement of the Connector helix. Our results suggest that the spatial orientation of the Connector helix, Platform and PAZ relative to the RNase III domains is crucial for measuring dsRNA of defined length. The modeled complexes provide information about interactions that may contribute to the relative orientations of these domains and to dsRNA binding. All these information can be helpful for understanding the mechanism of AtDCL4-mediated dsRNA recognition and binding, to produce small RNA of specific size.     

Modeling and simulation of morphology variation during the solidification of polymer melts with amorphous and semi‐crystalline phases

The solidification of polymer melts in practical processing such as extrusion, injection molding and blow molding can significantly influence the inner structure and performance of final products. The investigation of its mechanism has both scientific and industrial interests. In the study, the three‐dimensional mathematical model is developed for the simulation of morphology variation in the solidification of polymer melts with amorphous and semi‐crystalline phases. The amorphous phase is simulated as the finite extensible nonlinear elastic dumbbell with a peterlin closure approximation (FENE‐P) fluid and the semi‐crystalline phase is approximated as rigid rods that grow and oriented in the flow field. The model of amorphous phase and semi‐crystalline phase are coupled through the stress and momentum balance and the feedback of crystallinity to the system relaxation time. The evolution of crystallization kinetics process are described by using a set of Schneider equation that discriminating the relative roles of the thermal and the flow effect on the crystallization behavior. With the standard Galerkin formulation adopted as basic computational framework, the discrete elastic viscous stress splitting algorithm in cooperating with the streamline upwinding approach serves as a relatively robust numerical scheme by using penalty finite element–finite difference simulation with a decoupled solving algorithm. The proposed mathematical model and numerical method have been successfully applied to the investigation of solidification of polymer melts in the extrusion process. The variations of orientation and crystallization morphology during the solidification process are further discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular dynamics simulation studies suggests unconventional roles of non-secretary laccases from enteropathogenic gut bacteria and Cryptococcus neoformans serotype D

Laccase in Cryptococcus neoformans is covalently linked to the carbohydrate moiety of the cell wall, which allows it to get access to the different substrates for catalyzing their oxidation and therefore plays a vital role in the virulence. The laccase gene (3.0 kb) from C. neoformans serotype D was amplified, cloned and sequenced for protein modeling, docking and simulation studies. The three dimensional homology models of laccase protein from C. neoformans and other pathogenic gut bacteria were docked with selected biomolecules like prostaglandins (PG), membrane phospholipids, neurotransmitters (serotonin) using GOLD software. The GOLDscore values of laccase from C. neoformans docked with prostaglandinH₂ (59.76), prostaglandinG₂ (59.45), prostaglandinE₂ (60.99), phosphatidylinositol (54.95), phosphatidylcholine (46.26), phosphatidylserine (55.26), arachidonic acid (53.08) and serotonin (46.22) were similar to the laccase from enteropathogenic bacteria but showed a better binding affinity as compared to that of the non-pathogenic bacteria (e.g. Bacillus safensis, Bacillus pumilus and Bacillus subtilis). The RMSD of MD simulation study done for 25 ns using laccase protein from C. neoformans complexed with phosphatidylcholine was found to be highly stable, followed by the laccase-PGE₂ and laccase-serotonin complexes. Furthermore, the binding free energy results were found to support the docking and MD simulation results. The present study implies that few candidate ligands can be intermediate substrate in the catalysis of microbial laccases, which can further play some crucial role in the cell signaling and pathogenesis of enteropathogenic gut micro flora and C. neoformans.